Defects in heme biosynthesis disrupt iron homeostasis, leading to malfunction in erythropoiesis. Ferrochelatase, the only known human chelatase in the recently recognized chelatase family of enzymes, catalyzes the last step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin. Distortion of porphyrin, following its binding to ferrochelatase, is a crucial step in the catalytic mechanism of this enzyme. Analysis of structural and kinetic data pertinent to the mechanism of chelatases led the P.I. and collaborators to propose that the chelatase-induced distortion of porphyrin substrate not only enhances the reaction rate by decreasing the activation energy of the reaction but also modulates which divalent metal ion is incorporated into the porphyrin ring. In addition, preliminary results show that only a few mutations in the ferrochelatase scaffold are sufficient to alter ferrochelatase towards other metal chelatase activities. The P.I. proposes to use the ferrochelatase scaffold to assess how metal ion selectivity arises within the metal chelatase family. The hypothesis to be addressed is: Chelatases, by differentially distorting the porphyrin substrate, modulate which metal ion is incorporated into the porphyrin ring. To test this hypothesis, the following Specific Aims are proposed: 1. Define the mode and degree of porphyrin distortion induced by murine ferrochelatase and directly- evolved chelatase variants exhibiting different metal ion specificities and enzymatic activities. 2. Assess the molecular and structural determinants for metal ion selectivity and enzymatic activity of ferrochelatase and evolved variants. 3. Optimize metal ion selectivity of evolved chelatase variants. A combination of experimental approaches ranging from construction of focused metal chelatase libraries to the characterization of the mode of porphyrin distortion, metal-ion coordination geometry and kinetic properties will allow us to produce and analyze variants with subtle differences in the scaffold but different metal ion selectivities and chelatase activities. These findings will improve our understanding of heme biosynthesis and iron homeostasis and provide interpretations at a molecular level of erythropoietic disorders, and consequently a rational approach for their prevention, diagnosis and therapy.